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Creative Data Solutions (CDS) is a Vanderbilt Shared Resource and has extensive experience in providing effective and robust solutions to challenges pertaining to research data using modern informatics and bioinformatics approaches.

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Expression and purification of functional human 17 alpha-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. Use of this system for study of a novel form of combined 17 alpha-hydroxylase/17,20-lyase deficiency.

Enzymatically active human 17 alpha-hydroxylase cytochrome P450 (P450c17) has been expressed in and purified from Escherichia coli. The cDNA containing modifications within the amino-terminal eight codons which are favorable for expression in E. coli, as well as codons for 4 histidine residues at the carboxyl terminus, was placed in the pCWori+ expression vector. The modified human P450c17 was detected spectrophotometrically (400 nmol of P450c17/liter culture) and was found to be integrated into E. coli membranes. This previously inaccessible human P450 was purified to electrophoretic homogeneity (10.7 nmol of P450/mg) from solubilized bacterial membranes using two sequential chromatographic steps, nickel nitrilotriacetate followed by hydroxylapatite. The expected enzymatic activities of human P450c17 were reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase, giving turnover numbers of 8.0 nmol/min/nmol P450 for pregnenolone, 6.5 nmol/min/nmol P450 for progesterone, 0.06 nmol/min/nmol P450 for 17 alpha-hydroxypregnenolone, and no detectable activity for 17 alpha-hydroxyprogesterone. This system was utilized to study the molecular basis of a novel form of combined 17 alpha-hydroxylase, 17,20-lyase deficiency resulting from compound heterozygous mutations, a missense point mutation Tyr64(TAT)--> Ser (TCT), and an Ile112 duplication (ATCATC). Upon expression of these mutant proteins in E. coli, the Tyr64 mutant has 15% of the wild type 17 alpha-hydroxylase activity, whereas the Ile112 duplication shows no activity, results consistent with the observed clinical phenotype.